Impaired control of circulating blood glucose levels is the hallmark of diabetes. Blood glucose in a non-enzymatic statistical process may attach to the lysine residues of polypeptides thereby leading to glycated polypeptides. Glycation is frequently observed for proteins have a long half life. The protein most frequently used to assess long-term control of circulating blood glucose levels is hemoglobin.
There are numerous methods for determining glycated hemoglobin. These can be basically divided into three groups depending on the way in which glycated and non-glycated protein components are separated and quantified (Goldstein, D. E. et al., Clin. Chem. 32 Suppl. 10 (1986) B64-B70).
The first group consists of physicochemical methods based on the utilization of charge differences. These include the HPLC determination with cation exchanger columns such as Diamat, MonoS and PolyCat A (Bisse method) which are the most frequently used methods in clinical chemistry. In the case of glycated hemoglobin the quantitative evaluation is usually carried out by a relative measurement of the HbA1c signal in relation to the total amount of Hb (% HbA1c).
Methods in the second group are those which utilize the different chemical reactivity of glycated and non-glycated protein. These include the thiobarbituric acid method in which for example the glucose bound to hemoglobin is converted into a yellow dye and measured photometrically and also affinity chromatography methods in which complex formation between the vicinal diol groups of the sugar residue and a boric acid group that is bound covalently to a support is used to separate glycated and non-glycated hemoglobin. The separated substance classes are quantified photometrically and the relative amount of glycated hemoglobin is calculated or, in the case of the thiobarbituric acid method quantified, as an absolute determination by calibration with suitable standard materials.
Thirdly, immunological methods may be mentioned. Specific antibodies are used in immunological methods. These recognize for example the structural unit at the N-terminal end of the β chain of the glycated hemoglobin molecule which is typical for HbA1c (e.g., TINA-QUANT HbA1c, Roche Diagnostics GmbH, Germany). In the immunological methods the absolute content of both HbA1c and HbA0, respectively, is determined. This necessitates the use of calibrators which have been assigned a target concentration by an independent method. The relative content of for example HbA1c cannot be obtained by a direct measurement.
HbA1c is the major glycohemoglobin species in human blood. It has been used for almost 20 years for long-term assessment of glycemic control in diabetic patients. The comprehensive Diabetes Control and Complications Trial (DCCT) has provided ample evidence that microvascular complications such as retinopathy, nephropathy and neuropathy are directly related to the degree of hyperglycemia in patients with insulin-dependent diabetes (IDDM), and has proved that the measurement of HbA1c in blood is an excellent tool for long-term monitoring of the glycemic state of diabetic patients (The Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus, Nathan et al., N. Engl. J. Med 329 (1993) 977-986: Santiago, J. V., Diabetes 42 (1993) 1549-1554; Benjamin, J. and Sacks, D. B., Clin. Chem. 40 (1994) 683-687; and Goldstein, D. et al., Clin. Chem. 40 (1994) 1637-1640). The DCCT study has also clearly demonstrated the need for reliable and reproducible measurements of HbA1c and HbA0—the normal nonglycated hemoglobin, respectively.
The known methods are, however, associated with a number of disadvantages. Thus some of the physicochemical methods have a very poor selectivity since the measured signals of glycated protein are overlapped by the non-glycated variants The shapes of the chromatographic peaks are often asymmetrical and difficult to integrate. The cation exchanger columns that are used are susceptible to small changes in the working conditions and to contamination. Due to the poor selectivity there is a high risk of measuring values that are too high (false positive values).
In the case of chemical methods it is difficult to standardize the procedure and interferences by other components containing sugar can only be avoided with a large amount of effort. It is not possible to differentiate between for example HbA1c and other glycated hemoglobin variants.
The immunological methods are characterized by a very high selectivity towards glycated protein variants. However, the quality of the results depends on the quality of the standard used for calibration. Suitable primary standards in an optimal quality are not at present available, in particular for HbA1c. Information on matrix dependencies cannot be obtained due to a lack of a suitable reference method. In this case false positive values are also frequently obtained (see for example Tiran, A. et al., J. Clin. Lab. Anal. 8 (1994) 128-134).
Kobold U., et al. (U.S. Pat. No. 5,631,140) describe a method for the detection of glycated proteins like hemoglobin that is based on the proteolytic digestion of proteins comprised in that sample. Detection of peptidic fragments is thereafter performed by high performance liquid chromatography (HPLC) and mass spectroscopy (MS). Recently, Jeppsson, J.-O. et al., Clin. Chem. Lab. Med. 40 (2002) 78-89 reported on a reference method for measurement of HbA1c that has been approved by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). This method is based on the digestion of hemoglobin. Both the glycated and the non-glycated forms are enzymatically digested and the N-terminal peptide fragments of both forms of hemoglobin are quantified by HPLC-MS. In the enzymatic cleavage of hemoglobin endoproteinase Glu-C sequencing grade from Boehringer Mannheim, Mannheim, Germany (Id. no. 1047817) was used. To achieve complete digestion of hemoglobin an overnight digestion for e.g., 18 hours or a digestion with trypsin for 2 hours, respectively, is proposed.
Trypsin cleaves peptide bonds with lysine and arginine at the C-terminal side of the cleavage site, i.e., it cleaves between amino acids eight and nine of hemoglobin. Jeppsson et al., supra, were able to show that the lysine residues at position eight in the b-chain may also be glycated in samples with elevated HbA1c levels but not in those with normal levels. Thus using trypsin to release N-terminal octapeptides for quantification purpose would include the risk of getting doubly glycated octapeptides or singly glycated octapeptides glycated at the Lys-1 or at the Lys-8 position, respectively. Therefore these researchers concluded that trypsin cleavage was not usable. Endoproteinase Glu-C cleaves the N-terminal part of the b-chain between the two glutamic acid residues at positions six and seven. The resulting fragments contain only a single glycation site at the N-terminal valine and can thus be used to separate HbA1c. The actual cleaving site is easily exposed to the enzyme under mild denaturing conditions at pH 4.0. Complete denaturation prior to digestion exposes additional substrates to the enzyme and yields a more complex peptide mixture. By using the modern multidimensional analytical techniques of on-line HPLC and electrospray-mass spectrometry or the off-line system of HPLC and capillary electrophoresis the two β-N-terminal hexapeptides of HbA1c and HbA0 could be separated and quantified with the necessary analytical performance. Analyzing the mixture of peptide fragments resulting from the endoproteinase Glu-C digestion of whole blood samples, they obtained a high specificity and sensitivity in the measurement of HbA1c.
Some proteolytic enzymes lead to peptidic fragments that are likely to be generated at different rates. This in turn results either in a high variation of the concentration measured or in a rather long incubation time. A reagent used in the digestion of hemoglobin in order to assess the fraction of glycated hemoglobin should allow for both a rapid formation as well as for a stable formation of the desired peptidic fragments.
As will be appreciated, long digestion times go to the expense of sample throughput and are costly. For clinical routine a reagent ensuring the rapid digestion of hemoglobin and at the same time allowing for a precise quantization of e.g., HbA1c and HbA0 would be highly desirable. However, it appears that the methods for detection of hemoglobin that are based on the digestion of hemoglobin even nowadays require either quite long incubation times and/or do not lead to stable peptidic fragments.
It has now been found and established that a proteolytic reagent as disclosed below and as described in the appending claims can be provided that is very useful in the digestion of proteins and thereby e.g., leads to a rapid digestion of glycated and non-glycated hemoglobin.